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Antimicrobial Efficacy and Biofilm Testing

How do biofilm-associated infections impact the efficacy of antimicrobial products?

iFyber specializes in antimicrobial efficacy and biofilm testing solutions to verify the efficacy of antimicrobial medical devices and drugs. These include but are not limited to antimicrobial dressings, bio-engineered tissue products, biomaterials, bioactive dressings, and novel anti-infectives.

Biofilms pose a major clinical threat. Current US National Institutes of Health estimates suggest biofilms account for over 80% of all microbial infections. Given the relatively recent understanding of biofilms, biofilm testing is becoming an increasingly common addition to the screening profile of new antimicrobial technologies.

iFyber offers a menu of assays and biofilm testing solutions that assess biofilm prevention and eradication efficacy, in addition to custom biofilm test models often needed for developing technologies to combat resistant biofilm infections. In addition to our standard and custom biofilm testing program, iFyber offers various antimicrobial efficacy testing options for planktonic (free-floating) microorganisms.

By taking a multi-method biofilm testing approach, treatments can be evaluated for their ability to both prevent the formation of biofilms and eradicate existing biofilms. Whether you need biofilm susceptibility testing or to evaluate the antimicrobial efficacy of a product, you can get the data and answers you need with iFyber.

iFyber offers a menu of biofilm tests and assays that assess biofilm prevention and eradication efficacy, in addition to custom biofilm test models needed for developing technologies to combat resistant biofilm infections.

By taking a multi-method biofilm testing approach, treatments can be evaluated for their ability to both prevent the formation of biofilms and eradicate existing biofilms. Get the data and answers you need with iFyber.

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What are biofilms?

Complex communities of microbes protected within a matrix of sugars, proteins, and extracellular DNA.
Biofilms can form on a range of surfaces, including medical devices and human tissue systems.
Compared to free-floating (planktonic) forms of microorganisms, biofilm-associated microorganisms are more difficult to kill due largely to the presence of this protective matrix and a reduction of metabolic activity.

A graphic showing Biofilm, Highlighting a need for Antimicrobial efficacy and biofilm testing

Antimicrobial Efficacy and Biofilm Testing Services:

Antimicrobial Efficacy Testing:

MIC/MBC Testing

Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism after overnight incubation. This measure is important in determining the in vitro activity of new antimicrobials. The MIC is generally determined using a broth dilution assay, which involves creating serial dilutions of the antimicrobial in a liquid growth medium inoculated with a standardized number of microorganisms. After incubation, the lowest concentration that prevents the medium from becoming turbid is identified as the MIC.

The MIC test is essential for various reasons. Primarily, it helps in identifying the appropriate concentration of an antimicrobial required for effective treatment, ensuring that the final product has the necessary potency to combat infections. The Clinical and Laboratory Standards Institute (CLSI) has established guidelines, such as the CLSI M07-A9, which are considered the gold standard for conducting MIC tests. However, iFyber has the experience to fully customize experimental designs beyond standard procedures.

Once the MIC has been determined, the next step often involves finding the Minimum Bactericidal Concentration (MBC), which is the lowest concentration of an antimicrobial that kills the microorganism. The MBC is determined by taking samples from the MIC test wells that showed no growth and culturing them on antimicrobial-free agar plates. These plates are then incubated for 24 hours at 37°C ± 2°C. The MBC is identified as the lowest concentration at which no bacterial colonies grow on the agar, indicating that the antimicrobial concentration is sufficient to kill the bacteria rather than merely inhibiting their growth.

Determining the MBC is important for understanding the bactericidal capabilities of an antimicrobial agent. While the MIC indicates the potential of an antimicrobial to inhibit growth, the MBC provides a more comprehensive picture of its killing power.

Time-Kill Assessment

Time-Kill assessment is a dynamic method used to evaluate the efficacy of antimicrobial agents over time. This method involves exposing a microbial population to an antimicrobial agent and then measuring the reduction in viable microorganisms at various time intervals. Typically, the test is performed by inoculating a microbial culture in a growth medium containing the antimicrobial agent, followed by sampling at specific time points. These samples are then plated on agar to determine the number of surviving colonies and calculate microbial reduction.

The importance of Time-Kill assessment lies in its ability to provide detailed information on the kinetics of antimicrobial activity. Unlike static methods such as Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), which offer single-point measurements, Time-Kill studies capture the dynamic interaction between the antimicrobial agent and the microorganisms over time. This is particularly crucial for understanding how quickly an antimicrobial can reduce microbial load, which is vital for treating acute infections where rapid bacterial eradication is necessary. Furthermore, Time-Kill assays can help identify potential synergistic effects when using combination therapies, guiding the development of more effective treatment regimens.

Zone of Inhibition (ZOI)

The Zone of Inhibition is a fundamental qualitative test widely used in the medical device and pharmaceutical industries. Researchers developing antimicrobial textiles, surfaces, and liquids use this test for its simplicity and speed in measuring and comparing levels of inhibitory activity. In this method, approximately one million cells from a single strain are spread over an agar plate and incubated with the antimicrobial product. If the bacterial or fungal strain is susceptible to the antimicrobial agent, a clear zone appears on the agar plate; if resistant, no zone is observed. This test is most effective when the antimicrobial can leach out of the material, commonly including leachable antimicrobials embedded in plastics, textiles, solids, surfaces, liquids, gels, and antibiotic solutions.

The significance of the Zone of Inhibition test lies in its simplicity, cost-effectiveness, and ability to deliver quick preliminary results about an antimicrobial's potency. Additionally, it is a valuable tool for monitoring antibiotic resistance, as changes in the size of the Zone of Inhibition over time can indicate shifts in bacterial susceptibility. By providing a straightforward and visual assessment of antimicrobial activity, the Zone of Inhibition test is crucial for the development and implementation of effective antimicrobial therapies.

AATCC Test Method 100

Test Method 100 evaluates the effectiveness of antimicrobial finishes on textile materials. The test articles are directly inoculated with the test organism and incubated at variable times depending on the application (typically at least 24 hours). The test articles are then placed in neutralizing solution and the microbes are recovered and enumerated.

Test Method 100 is the industry standard for evaluating the antimicrobial efficacy of textiles. It provides a quantitative assessment of growth inhibition or microbial killing within the wound dressing material. The results can be used for 510(k) filings or other product claims for marketing purposes. Learn more about the use of the AATCC test method 100.

Download the AATCC 100 Service Sheet

Biofilm Testing:

In-vitro: Bottom-of-the-Plate (BOTP) Assay

Biofilm susceptibility testing is critical in the medical device industries because biofilms can form on implants, catheters, and other medical devices, leading to persistent infections. Biofilms are highly resistant to standard antimicrobial treatments, making infections difficult to eradicate. Testing ensures that medical devices and pharmaceutical products can effectively prevent and eliminate biofilms, improving patient outcomes and reducing infection-related complications.

The Minimum Biofilm Eradication Concentration (MBEC) assay is a commonly used in vitro high throughput screening method to assess the efficacy of antimicrobials against biofilms formed by various microorganisms. However, the MBEC assay has limitations, such as its suitability for only a limited range of microorganisms and high variability in results. To address these challenges, iFyber has developed an alternative model known as the Bottom-of-the-Plate (BOTP) assay. In this model, diverse microorganisms can form mature biofilms at the bottom of 96-well plates. The BOTP assay serves as a high throughput alternative to the MBEC assay, offering less variability and more reliable data. It is well-suited for evaluating the prevention and eradication of biofilms by a variety of antimicrobial products.

Ex-vivo: Porcine Dermal Model (PDM)

This custom assay is a robust model of a mature bacterial biofilm, which are a hallmark of many bacterial infections that occur in soft tissues. This model isolates the biofilm component of these infections through the growth of a mature biofilm within a natural porcine dermal matrix. The resulting biofilm (typically grown over 72 hours) exhibits high tolerance to many antibiotics and, importantly, incorporates a relevant substrate when assessing product efficacy in the presence of soft tissue. iFyber has extensive experience adapting this model to our clients needs to test for antimicrobial and anti-biofilm activity against a model for mature biofilm exhibiting tolerance to high doses of antibiotics. In this model, tissue explants are created, wounded, and subsequently infected to enable high throughput screening using a number of assay formats (prevention, eradication, dispersal, synergy).

The characteristics of biofilms grown on non-biological substrates can differ from what occurs in soft tissue. When looking for antimicrobial solutions to eradicate biofilms from wounds in particular, it can be informative to use an assay that closely approximates the wound environment. Porcine skin is physiologically very similar to that of human skin. Using skin as both the substrate for attachment, and the primary source of nutrition, allows for the development of a biofilm that mimics the characteristics of biofilms found in human wounds. This ex vivo model allows for a high throughput screening of compounds, prototype formulations, solid wound dressings, etc., to complement/augment more costly animal studies.

Histology and Imaging

As a complement to our antimicrobial offerings, iFyber routinely performs histological analyses and microscopy techniques for evaluating biofilm prevention and eradication both in vitro and ex vivo models. Visualization of microorganisms can be performed through different staining techniques such as Gram staining and LIVE/DEAD staining. A variety of microscopy techniques can be utilized to visualize the planktonic and biofilm microorganisms, such as, bright field, confocal, or SEM. These techniques allow for qualitative visual evidence of the effects of anti-biofilm treatments on the structure and morphology of underlying tissues and the prevention or eradication of infiltrating biofilm. Data derived from these models can aid the development of effective device- and drug-based wound care treatments.

Learn more about iFyber's Histology and Imaging offerings.

Infections Associated with Biofilms:

Chronic Infected Wounds
Catheter-associated urinary Tract Infections (CAUTI)
Central line-associated and catheter-related bloodstream Infections (CLABSI and CRBI)
Acute Bacterial Skin and Skin Suture Infection (ABSSSI)
Ventilator-associated Pneumonia (VAP)
Acute Otitis Media (Ear Infection)
Acute Vulgaris
Gingivitis
Cystic Fibrosis Lung Infection
And more

Examples of Relevant Materials for Biofilm Testing:

New antibiotics/biocides
Coatings
Nanomaterials
Wound dressings
Gels and ointments
Wound washes
Coated natural tissue matrices
Coated medical devices
And more

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Why Choose iFyber forAntimicrobial Efficacy and Biofilm Testing:

Specialized Expertise and Investment in Your Research Success. iFyber focuses on specific research questions. This allows us to tailor our approach to your unique needs, ensuring the data we generate is directly relevant to your goals.
Get Information, Not Just Data: We’ll help guide you on what scientific services you need, choose methods, and design experiments to provide actionable information. Then once the experiment is complete, we’ll provide insight and context on what it means, the next steps, and more.
Clear Communication and Collaboration. We believe in close collaboration with our clients, keeping you informed throughout the process and ensuring you fully understand the data and its implications for your research. High-touch, accessible support from a trusted team.

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Ready to elevate your research? Discover precision, expertise, and collaboration with iFyber. Contact us now to discuss your antimicrobial and biofilm testing needs.

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